Characterization of the amp genes involved in the regulation of beta-lactamase expression in Pseudomonas aeruginosa

Antibiotic resistance, production of alginate and virulence factors, and altered host immune responses are the hallmarks of chronic Pseudomonas aeruginosa infection. Failure of antibiotic therapy has been attributed to the emergence of P. aeruginosa strains that produce β-lactamase constitutively. In Enterobacteriaceae, β-lactamase induction involves four genes with known functions: ampC, ampR, ampD, and ampG, encoding the enzyme, transcriptional regulator, amidase and permease, respectively. In addition to all these amp genes, P. aeruginosa possesses two ampG paralogs, designated ampG and ampP. In this study, P. aeruginosa ampC, ampR, ampG and ampP were analyzed. Inactivation of ampC in the prototypic PAO1 failed to abolish the β-lactamase activity leading to the discovery of P. aeruginosa oxacillinase PoxB. Cloning and expression of poxB in Escherichia coli confers β-lactam resistance. Both AmpC and PoxB contribute to P. aeruginosa resistance against a wide spectrum of β-lactam antibiotics. The expression of PoxB and AmpC is regulated by a LysR-type transcriptional regulator AmpR that up-regulates AmpC but down-regulates PoxB activities. Analyses of P. aeruginosa ampR mutant demonstrate that AmpR is a global regulator that modulates the expressions of Las and Rhl quorum sensing (QS) systems, and the production of pyocyanin, LasA protease and LasB elastase. Introduction of the ampR mutation into an alginate-producing strain reveals the presence of a complex co-regulatory network between antibiotic resistance, QS alginate and other virulence factor production. Using phoA and lacZ protein fusion analyses, AmpR, AmpG and AmpP were localized to the inner membrane with one, 16 and 10 transmembrane helices, respectively. AmpR has a cytoplasmic DNA-binding and a periplasmic substrate binding domains. AmpG and AmpP are essential for the maximal expression of β-lactamase. Analysis of the murein breakdown products suggests that AmpG exports UDP-N-acetylmuramyl-L-alanine-γ-D-glutamate-meso-diaminopimelic acid-D-alanine-D-alanine (UDP-MurNAc-pentapeptide), the corepressor of AmpR, whereas AmpP imports N-acetylglucosaminyl-beta-1,4-anhydro-N-acetylmuramic acid-Ala-γ-D-Glu-meso-diaminopimelic acid (GlcNAc-anhMurNAc-tripeptide) and GlcNAc-anhMurNAc-pentapeptide, the co-inducers of AmpR. This study reveals a complex interaction between the Amp proteins and murein breakdown products involved in P. aeruginosa β-lactamase induction. In summary, this dissertation takes us a little closer to understanding the P. aeruginosa complex co-regulatory mechanism in the development of β-lactam resistance and establishment of chronic infection. ^

Seasonal and spatial variation in the stable isotopic composition (δ18O and δD) of precipitation in south Florida

Precipitation data collected from five sites in south Florida indicate a strong seasonal and spatial variation in δ18O and δD, despite the relatively limited geographic coverage and low-lying elevation of each of the collection sites. Based upon the weighted-mean stable isotope values, the sites were classified as coastal Atlantic, inland, and lower Florida Keys. The coastal Atlantic sites had weighted-mean values of δ18O and δD of −2.86‰ and −12.8‰, respectively, and exhibited a seasonal variation with lower δ18O and δD values in the summer wet-season precipitation (δ18O = −3.38‰, δD = −16.5‰) as compared to the winter-time precipitation (δ18O = −1.66‰, δD = −3.2‰). The inland site was characterized as having the highest d-excess value (+13.3‰), signifying a contribution of evaporated Everglades surface water to the local atmospheric moisture. In spite of its lower latitude, the lower Keys site located at Long Key had the lowest weighted-mean stable isotope values (δ18O = −3.64‰, δD = −20.2‰) as well as the lowest d-excess value of (+8.8‰). The lower δD and δ18O values observed at the Long Key site reflect the combined effects of oceanic vapor source, fractionation due to local precipitation, and slower equilibration of the larger raindrops nucleated by a maritime aerosol. Very low δ18O and δD values (δ18O < −6‰, δD < −40‰) were observed just prior to the passage of hurricanes from the Gulf of Mexico as well as during cold fronts from the north-west. These results suggest that an oceanic vapor source region to the west, may be responsible for the extremely low δD and δ18O values observed during some tropical storms and cold fronts.